Biological substance detection method

ABSTRACT

The present invention provides a biological substance detection method for specifically detecting a biological substance from a pathological specimen, by which method, when immunostaining using a fluorescent label and staining for morphological observation using a staining agent for morphological observation are simultaneously performed, the results of fluorescence observation and immunostaining can be assessed properly even if the fluorescent label and/or the staining agent is/are deteriorated by irradiation with an excitation light. The biological substance detection method according to the present invention is characterized in that the brightness retention rate of an immunostained part is in a range of 80% to 120% in relation to the brightness retention rate of a part stained for morphological observation when the fluorescent label used for the immunostaining is observed.

CROSS REFERENCE TO RELATED APPLICATION

This is a divisional application of U.S. application Ser. No. 14/343,411filed on Mar. 7, 2014, which was a U.S. National Phase Application under35 U.S.C. 371 of PCT/JP2012/072496 filed on Sep. 4, 2012 which claimedthe priority of Japanese Patent Application No. 2011-197339 filed onSep. 9, 2011, the entire content of all three Applications are herebyincorporated by reference.

TECHNICAL FIELD

The present invention relates to a biological substance detectionmethod. More particularly, the present invention relates to tissuestaining in which a tissue is stained with a fluorescent label.

BACKGROUND ART

As a medical diagnosis, a pathological diagnosis is performed. Apathologist diagnoses a disease using a tissue section collected from ahuman body and informs a clinician of whether or not a therapy and/or asurgery is/are necessary. Based on the patient conditions andpathological diagnosis, a physician determines pharmacotherapeuticstrategies and a surgeon determines whether or not a surgery should beperformed.

In pathological diagnosis, it is a common practice to prepare a tissuesample by slicing a tissue specimen obtained by evisceration or needlebiopsy into a thickness of several micrometers or so and observe thetissue sample at a magnification under a light microscope so as toobtain various findings. In many cases, a specimen is prepared by fixinga collected tissue through dehydration and paraffin blocking, slicingthe resultant into a thickness of several micrometers and then removingthe paraffin. Here, since the specimen hardly absorbs or scatters anylight and is thus nearly colorless and transparent, it is usuallystained with a dye prior to being observed.

There have been proposed a variety of staining techniques. Inparticular, for tissue samples, hematoxylin-eosin staining (HE staining)using two dyes, hematoxylin and eosin, is typically used as staining forobserving the morphology of a specimen (Patent Literatures 1 and 2).Hematoxylin stains cell nuclei, calcareous parts, cartilaginous tissues,bacteria and mucus in livid to light blue, while eosin stains cytoplasm,interstitial tissues, various fibers, erythrocytes and keratinocyte inred to dark red. A pathologist makes a diagnosis based on morphologicalinformation and staining information, such as changes in the size andshape of cell nuclei and changes in the pattern as a tissue, in amicroscope image of a stained tissue sample. Examples of other stainingfor morphological observation include Papanicolaou staining (Papstaining) used for cytological diagnosis.

Further, in pathological diagnosis, immunological observation in whichmolecular target staining called immunostaining is performed forconfirming the expression of molecular information of a specimen andfunctional abnormalities such as abnormal expression of a gene or aprotein are diagnosed is performed. For immunostaining, for example, adye staining method using an enzyme (DAB staining) is employed. In DABstaining, an antibody modified with peroxidase, which is capable ofallowing DAB (diaminobenzidine) as a substrate to show a color, is usedto stain an antigen to be observed with the color and the amount of theantigen is determined by observing the stained antigen. Alternatively,fluorescent labeling may be employed. In fluorescent labeling, theamount of the subject antigen is determined by staining the antigen withan antibody modified with a fluorescent dye and observing the stainedantigen.

At present, attempts are being made to simultaneously performmorphological observation and immunological observation of a specimenand, for example, it has been tried to simultaneously perform HEstaining for morphological observation and DAB staining forimmunological observation (Patent Literature 3). However, since stainingwith an enzyme label, such as DAB staining, develops a color similar tothe color developed by HE staining and the colors developed by HEstaining and staining with an enzyme label cannot thus be easilydistinguished, there is a problem that such simultaneous observation isdifficult. In addition, in DAB staining, since the stainingconcentration is largely affected by the environmental conditions suchas temperature and time, there is a problem that estimation of theactual amount of an antibody or the like from the staining concentrationis difficult.

On another front, in pathological diagnosis, fluorescent labeling usinga fluorescent label is also performed. This method characteristicallyhas excellent quantitative capability as compared to DAB staining. Incases where pathological diagnosis and morphological observation aresimultaneously performed using a fluorescent label, there is a problemthat the results are likely to be affected by the fluorescence of thestaining agent used for tissue staining. As a countermeasure, forexample, a fluorescent dye which has peaks of excitation and emissionwavelengths in the infrared region and is thus not likely to be affectedby visible light can be used (Patent Literature 4). Alternatively, forexample, the excitation and emission wavelengths of a staining agent formorphological observation and those of a fluorescent label forimmunostaining can be shifted.

Meanwhile, it is known that dyes are easily deteriorated generally inthe short-wavelength side, particularly in the ultraviolet region.Considering the effect on deterioration of a staining agent and afluorescent label, it is desired that they be excited in the visibleregion rather than in the ultraviolet region.

As fluorescent labels, dyes, inorganic nanoparticles (that may also bereferred to as “semiconductor nanoparticle”, “quantum dot” or the like)and aggregates thereof are known to be utilized (Patent Document 5).Thereamong, inorganic nanoparticles are not suitable as theabove-described fluorescent label to be excited in the visible region;therefore, it is difficult to utilize inorganic nanoparticles. On theother hand, fluorescent dyes and aggregates thereof can be utilized asthe above-described fluorescent label to be excited in the visibleregion. It has been reported that, between a fluorescent dye and anaggregate thereof, the light-resistant performance is more improved bythe latter (Patent Document 6). Furthermore, between a dye and anaggregate thereof, the brightness of individual dye is higher in theaggregate.

CITATION LIST Patent Literatures

[Patent Literature 1] JP 2001-525580 A

[Patent Literature 2] JP 2009-115599 A

[Patent Literature 3] JP 2010-134195 A

[Patent Literature 4] WO 2008/006006

[Patent Literature 5] JP 2010-209314 A

[Patent Literature 6] JP 2008-147394 A

SUMMARY OF INVENTION Technical Problem

In cases where pathological diagnosis and morphological observation aresimultaneously performed using the above-described fluorescent label, aconfocal laser microscope or a fluorescence microscope is used forobservation of the fluorescent label. In fluorescence observation underthese microscopes, a stained section is irradiated with a high-intensityexcitation light. This excitation light gradually deterioratesfluorescent labels using a fluorescent dye or the like and stainingagents such as eosin and this has a great effect in fluorescenceobservation and assessment of immunostaining results. Ideally, neitherof a fluorescent label nor a staining agent is deteriorated; however,the reality is that they are deteriorated at a certain rate.

In view of the above, a main object of the present invention is toprovide a method by which the results of fluorescence observation andimmunostaining can be assessed properly even when a fluorescent labeland/or a staining agent is/are deteriorated by irradiation with anexcitation light as described above.

Solution to Problem

The present inventors discovered a biological substance detection methodfor specifically detecting a biological substance from a pathologicalspecimen, in which method, in cases where immunostaining using afluorescent label (particularly a fluorescent dye-containingnanoparticle) and staining for morphological observation using astaining agent for morphological observation are simultaneouslyperformed, assessments of the results of fluorescence observation andimmunostaining are hardly affected when the fluorescent label used forimmunostaining is observed and the brightness retention rate (ratio ofthe brightness at the time of the observation with respect to thebrightness at the initiation of irradiation with an excitation light) ofan immunostained part (sites that are immunostained with the fluorescentlabel) is found to be in a range of 80% to 120% in relation to thebrightness retention rate of apart stained for morphological observation(sites that are not immunostained with the fluorescent label but stainedwith the staining agent for morphological observation).

That is, the present invention encompasses the followings.

-   [1] A biological substance detection method for specifically    detecting a biological substance from a pathological specimen, the    method being characterized in that, in cases where immunostaining    using a fluorescent label and staining for morphological observation    using a staining agent for morphological observation are    simultaneously performed, the brightness retention rate of an    immunostained part is in a range of 80% to 120% in relation to the    brightness retention rate of a part stained for morphological    observation when the fluorescent label used for the immunostaining    is observed.-   [2] The biological substance detection method according to [1],    wherein the above-described fluorescent label is a fluorescent    dye-containing nanoparticle whose maximum excitation wavelength is    in a wavelength range of 550 to 620 nm and maximum emission    wavelength is in a wavelength range of 580 to 700 nm.-   [3] The biological substance detection method according to [2],    wherein the above-described fluorescent dye is a rhodamine-based dye    molecule or an aromatic ring-based dye molecule.-   [4] The biological substance detection method according to [3],    wherein the above-described fluorescent dye is perylene.-   [5] The biological substance detection method according to any one    of [2] to [4], wherein a parent material of the above-described    fluorescent dye-containing nanoparticle is any of polystyrene,    polyamide, polylactic acid, polyacrylonitrile, polyglycidyl    methacrylate, polymelamine, polyurea, polybenzoguanamine, polyfuran,    polyxylene, phenol resin, polysaccharide and silica.-   [6] The biological substance detection method according to any one    of [1] to [5], wherein the above-described staining agent has a    maximum excitation wavelength in a wavelength range of 450 nm to 550    nm.-   [7] The biological substance detection method according to [6],    wherein the above-described staining agent is eosin.

Advantageous Effects of Invention

According to the biological substance detection method of the presentinvention, the results of fluorescence observation and immunostainingcan be assessed properly even when a fluorescent label and/or a stainingagent is deteriorated.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the fluorescence spectrum (excitation wavelength=520 nm)and the excitation spectrum (fluorescence wavelength=540 nm) of eosin.

FIG. 2 is a graph showing the brightness retention rate of an eosin part(80% in all cases) and that of the respective labeled parts in Examples1 to 11 (samples 1-1 to 1-11) and Comparative Examples 1 to 4 (samples2-1 to 2-4).

DESCRIPTION OF EMBODIMENTS

The mode for carrying out the invention will now be described; however,the present invention is not restricted thereto.

The biological substance detection method according to a typicalembodiment of the present invention is a method of specificallydetecting a biological substance from a pathological specimen, whichbasically comprises: (1) the step of immunostaining a pathologicalspecimen with a fluorescent label; (2) the step of staining thepathological specimen for morphological observation with a stainingagent for morphological observation; and (3) the step of irradiating thethus stained pathological specimen with an excitation light to allowfluorescence to be emitted for detection of a biological substance fromthe pathological specimen. In addition, the biological substancedetection method may also comprise a deparaffinization step, anactivation treatment step and/or the like in the same manner as in anordinary biological substance detection method.

In the present invention, particularly in (1) the step of immunostaininga pathological specimen, it is expected that, as the fluorescent label,a fluorescent dye-containing nanoparticle whose excitation wavelength isin a range different from the excitation wavelength range of a stainingagent for morphological observation will be employed. However, in thepresent invention, the fluorescent label for immunostaining is notrestricted to such a fluorescent dye-containing nanoparticle and it isalso possible to use other fluorescent label which can satisfy acondition that, when observed, the brightness retention rate of animmunostained part is in a range of 80% to 120% in relation to thebrightness retention rate of a part stained for morphologicalobservation.

It is noted here that whichever (1) the step of immunostaining or (2)the step of staining for morphological observation may be performedfirst and the order (sequence) of these steps is not a matter ofimportance. Regardless of the order of the immunostaining and thestaining for morphological observation, “immunostaining using afluorescent label and staining for morphological observation using astaining agent for morphological observation are simultaneouslyperformed”.

The details of the staining agent for morphological observation, thefluorescent label for immunostaining (fluorescent dye-containingnanoparticle), the staining for morphological observation, theimmunostaining and the like will now be described below.

[Staining Agent for Morphological Observation]

As the staining agent for morphological observation, hematoxylin andeosin are particularly suitably used; however, the staining agent formorphological observation is not restricted thereto. Any dye may be usedas long as it is capable of staining cytoplasm and the like in the samemanner as eosin and has excitation and emission wavelengths that aresimilar to those of eosin in the visible region. For example, orange G,eosin Y and light green SFY, which are used in Papanicolaou staining(Pap staining) performed for cytological diagnosis, are also dyes havingexcitation and emission wavelengths that are similar to those of eosinin the visible region. As for hematoxylin as well, any dye may be usedas long as it is capable of staining cell nuclei in the same manner ashematoxylin and has excitation and emission wavelengths that are similarto those of hematoxylin in the visible region.

[Fluorescent Label for Immunostaining]

In the present invention, the fluorescent label for immunostaining isone which can achieve a condition that, when the fluorescent label usedfor immunostaining is observed, the brightness retention rate of animmunostained part is in a range of 80% to 120% in relation to thebrightness retention rate of a part stained for morphologicalobservation. That is, as the fluorescent label for immunostaining, onewhose reduction in the brightness retention rate caused by irradiationwith an excitation light can be synchronized within a certain range witha reduction in the brightness retention rate of a staining agent formorphological observation (which is represented by hematoxylin andeosin) to be used in combination is employed.

Further, eosin used in HE staining, which is one of the stainingtechniques used for morphological observation, emits fluorescencedepending on the microscopy conditions. Since the absorption wavelengthof eosin overlaps with the excitation wavelengths of many fluorescentlabels, the fluorescence emitted by eosin used in the staining maypotentially interfere with the observation of a fluorescent label. Thefluorescence spectrum (excitation wavelength=520 nm) and the excitationspectrum (fluorescence wavelength=540 nm) of eosin are shown in FIG. 1.From the excitation spectrum, it is seen that eosin is efficientlyexcited in a wavelength range of longer than 450 nm to shorter than 550nm. Therefore, in the present invention, it is preferred that thefluorescent label for immunostaining be one which has a maximumexcitation wavelength outside this wavelength range, that is, in eithera wavelength range of 350 to 450 nm or a long wavelength range of 550 nmor longer. It is noted here, however, that the maximum excitationwavelength is more preferably 620 nm or shorter, considering the balancewith the fluorescence wavelength. From the standpoint of reducing theeffect of excitation light on the staining agent as much as possible, itis more preferred to use a fluorescent label having a maximum excitationwavelength in a wavelength range of 550 to 620 nm than using afluorescent label having a maximum excitation wavelength in a wavelengthrange of 350 to 450 nm. Meanwhile, considering the balance with theeffects of absorption and emission by eosin and the tissue intrinsicfluorescence, it is preferred that the fluorescent label have a maximumemission wavelength in the long-wavelength side of 580 nm or longer.Further, since the fluorescent label is required to be visuallyconfirmable when observed under a fluorescence microscope, it is alsopreferred that the fluorescent label have a maximum emission wavelengthin the short-wavelength side of 700 nm or shorter.

[Fluorescent Dye-containing Nanoparticle]

From the standpoint of the ratio of the signal value against thefluorescence of eosin and the intrinsic fluorescence of cells, which arenoises, the higher the brightness of the fluorescent label, the morepreferred it is. Therefore, in the present invention, a fluorescentdye-containing nanoparticle having a higher brightness than afluorescent dye is suitably used as the fluorescent label.

The term “fluorescent dye-containing nanoparticle” refers to anano-sized particle having a structure in which plural fluorescent dyesare encapsulated in a particle (parent material) made of an organic orinorganic material. The fluorescent dye-containing nanoparticle used inthe present invention can be prepared by a known method by selecting, asraw materials, fluorescent dyes suitable for controlling the brightnessretention rate of an immunostained part to be in a range of 80% to 120%in relation to the brightness retention rate of a part stained formorphological observation as well as an organic or inorganic materialfor forming a particle.

Examples of the organic or inorganic material for forming a particleinclude those into which fluorescent dyes can be encapsulated, such aspolystyrene, polyamide, polylactic acid, polyacrylonitrile, polyglycidylmethacrylate, polymelamine, polyurea, polybenzoguanamine, polyfuran,polyxylene, phenol resins, polysaccharides and silica. By encapsulatingfluorescent dyes into such a particle, a fluorescent dye-containingnanoparticle in which deterioration caused by irradiation with anexcitation light is less likely to occur as compared to a case where thefluorescent dyes are used by themselves (high light resistance) and thebrightness retention rate of an immunostained part can be adjusted in arange of 80% to 120% in relation to the brightness retention rate of apart stained for morphological observation can be produced. For example,hydrophobic compounds such as polystyrene, polymelamine and silica arepreferred as the parent material of a highly light-resistant,fluorescent dye-containing nanoparticle.

Meanwhile, it is preferred that the fluorescent dyes to be encapsulatedbe those which are excited in a wavelength range of 350 to 450 nm or ina long wavelength range of 550 nm or longer such that their excitationwavelengths do not overlap with the absorption wavelength of eosin,which is a representative staining agent for morphological observation.

Such fluorescent dyes can be selected from, for example, rhodamine-baseddye molecules, squarylium-based dye molecules, cyanine-based dyemolecules, aromatic ring-based dye molecules, oxazine-based dyemolecules, carbopyronine-based dye molecules and pyrromethene-based dyemolecules. Alternatively, the fluorescent dyes can also be selectedfrom, for example, Alexa Fluor-based dye molecules (registeredtrademark, manufactured by Invitrogen), BODIPY-based dye molecules(registered trademark, manufactured by Invitrogen), Cy-based dyemolecules (registered trademark, manufactured by GE Healthcare),DY-based dye molecules (registered trademark, Dyomics GmbH),HiLyte-based dye molecules (registered trademark, manufactured byAnaSpec Inc.), DyLight-based dye molecules (registered trademark,manufactured by Thermo Fisher Scientific K.K.), ATTO-based dye molecules(registered trademark, manufactured by ATTO-TEC GmbH) and MFP-based dyemolecules (registered trademark, manufactured by Mobitec Co., Ltd.). Thegeneric names of these dye molecules are designated based on the mainstructure (skeleton) or the registered trademark of the respectivecompounds; therefore, those of ordinary skill in the art can properlyunderstand the scope of fluorescent dyes belonging to the respectivegeneric names without having to bear undue trial and error.

Specific examples of the rhodamine-based dye molecules include5-carboxy-rhodamine, 6-carboxy-rhodamine, 5,6-dicarboxy-rhodamine,rhodamine 6G, tetramethyl rhodamine, X-rhodamine, Texas Red, SpectrumRed and LD700 PERCHLORATE.

Specific examples of the squarylium-based dye molecules include SRfluor680-carboxylate,1,3-Bis[4-(dimethylamino)-2-hydroxyphenyl]-2,4-dihydroxycyclobutenediyliumdihydroxide,bis,1,3-bis[4-(dimethylamino)phenyl]-2,4-dihydroxycyclobutenediyliumdihydroxide,bis,2-(4-(diethylamino)-2-hydroxyphenyl)-4-(4-(diethyliminio)-2-hydroxycyclohexa-2,5-dienylidene)-3-oxocyclobut-1-enolate,2-(4-(dibutylamino)-2-hydroxyphenyl)-4-(4-(dibutyliminio)-2-hydroxycyclohexa-2,5-dienylidene)-3-oxocyclobut-1-enolateand2-(8-hydroxy-1,1,7,7-tetramethyl-1,2,3,5,6,7-hexahydropyrido[3,2,1-ij]quinolin-9-yl)-4-(8-hydroxy-1,1,7,7-tetramethyl-2,3,6,7-tetrahydro-1H-pyrido[3,2,1-ij]quinolinium-9(5H)-ylidene)-3-oxocyclobut-1-enolate.

Specific examples of the cyanine-based dye molecules include1-butyl-2-[5-(1-butyl-1,3-dihydro-3,3-dimethyl-2H-indol-2-ylidene)-penta-1,3-dienyl]-3,3-dimethyl-3eiti-indoliumhexafluorophosphate,1-butyl-2-[5-(1-butyl-3,3-dimethyl-1,3-dihydro-indol-2-ylidene)-3-chloro-penta-1,3-dienyl]-3,3-dimethyl-3H-indoliumhexafluorophosphate and3-ethyl-2-[5-(3-ethyl-3H-benzothiazol-2-ylidene)-penta-1,3-dienyl]-benzothiazol-3-iumiodide.

Specific examples of the aromatic ring-based dye molecules includeN,N-bis-(2,6-diisopropylphenyl)-1,6,7,12-(4-tert-butylphenoxy)-perylen-3,4,9,10-tetracarbonaciddiimide,N,N′-bis(2,6-diisopropylphenyl)-1,6,7,12-tetraphenoxyperylene-3,4:9,10-tetracarboxdiimide,N,N′-bis(2,6-diisopropylphenyl)perylene-3,4,9,10-bis(dicarbimide),16,N,N′-bis(2,6-dimethylphenyl)perylene-3,4,9,10-tetracarboxylicdiimide,4,4′-[(8,16-dihydro-8,16-dioxodibenzo[a,j]perylene-2,10-diyl)dioxy]dibutyricacid, 2,10-dihydroxy-dibenzo[a,j]perylene-8,16-dione,2,10-bis(3-aminopropoxy)dibenzo[a,j]perylene-8,16-dione,3,3′-[(8,16-dihydro-8,16-dioxodibenzo[a,j]perylen-2,10-diyl)dioxy]dipropylamine,17-bis(octyloxy)anthra[9,1,2-cde-]benzo[rst]pentaphene-5-10-dione,octadecanoic acid,5,10-dihydro-5,10-dioxoanthra[9,1,2-cde]benzo[rst]pentaphene-16,17-diylesterand dihydroxydibenzanthrone.

Specific examples of the oxazine-based dye molecules include Cresylviolet, Oxazine 170, EVOblue 30 and Nile Blue.

Specific examples of the carbopyronine-based dye molecules includeCARBOPYRONIN 149.

Specific examples of the pyrromethene-based dye molecules includePYRROMETHENE 650.

Specific examples of the Alexa Fluor-based dye molecules include AlexaFluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, AlexaFluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, AlexaFluor 680, Alexa Fluor 700 and Alexa Fluor 750.

Specific examples of the BODIPY-based dye molecules include BODIPY FL,BODIPY TMR, BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650 and BODIPY650/665 (all of which are manufactured by Invitrogen).

Specific examples of the Cy-based dye molecules include Cy 3.5, Cy 5 andCy 5.5.

Specific examples of the DY-based dye molecules include DY-590, DY-610,DY-615, DY-630, DY-631, DY-632, DY-633 and DY-634.

Specific examples of the HiLyte-based dye molecules include HiLyte 594and HiLyteFluor TR.

Specific examples of the DyLight-based dye molecules include DyLight 594and DyLight 633.

Specific examples of the ATTO-based dye molecules include ATTO 590, ATTO610, ATTO 620, ATTO 633 and ATTO 655.

Specific examples of the MFP-based dye molecules include MFP 590 and MFP631.

Examples of other dye include C-phycocyanin, phycocyanin, APC(allophycocyanin), APC-XL and NorthernLights 637.

Further, examples of dyes also include derivatives of theabove-described dyes (those which can function as a fluorescent dye,such as known derivatives).

In the fluorescent dye-containing nanoparticle, any one of theabove-described fluorescent dyes may be encapsulated individually, or aplurality thereof may be encapsulated in combination.

For example, fluorescent dyes such as aromatic ring-based dye moleculesand rhodamine-based dye molecules are preferred because of theirrelatively high light resistance. Thereamong, perylenes belonging toaromatic ring-based dye molecules, particularly perylenediimide, arepreferred. Meanwhile, even when a fluorescent dye having a relativelylow light resistance is used, by selecting an appropriate parentmaterial, it is possible to produce a fluorescent dye-containingnanoparticle which satisfies a prescribed brightness retention ratecondition of the present invention.

The method of producing a fluorescent dye-containing nanoparticle is notparticularly restricted. For introduction of a dye (s) into a particle,any method such as a method of synthesizing a particle by binding a dyemolecule to a monomer that is a particle raw material or a method ofintroducing a dye to a particle by adsorption may be employed.

The average particle diameter of the fluorescent dye-containingnanoparticle is not particularly restricted; however, it is usually 10to 500 nm, preferably 50 to 200 nm. Further, the variation coefficientwhich indicates the variation in the particle size is also notparticularly restricted; however, it is usually about 20%. Here, theparticle size of a fluorescent dye-containing nanoparticle can bedetermined by taking an electron microscope image thereof using ascanning electron microscope (SEM), measuring the cross-sectional areaof the fluorescent dye-containing nanoparticle and then calculating theparticle size as the diameter of a circular area corresponding to themeasured value. With regard to the average particle size (averageparticle diameter) and the variation coefficient of a group offluorescent dye-containing nanoparticles, after measuring the particlesizes (particle diameters) of a sufficient number (for example, 1,000)of fluorescent dye-containing nanoparticles in the above-describedmanner, the average particle size is calculated as the arithmetic meanof the measured values and the variation coefficient is calculated bythe following equation: 100×standard deviation of particle size/averageparticle size.

[Step of Staining for Morphological Observation]

In the step of staining for morphological observation, particularly whenthe morphology of a tissue sample is observed, the above-describedhematoxylin-eosin staining (HE staining) using two dyes, hematoxylin andeosin, is typically used; however, in the present invention, thestaining for morphological observation is not restricted thereto.Examples of other staining for morphological observation includePapanicolaou staining (Pap staining) used for cytological diagnosis.

In HE staining, hematoxylin stains cell nuclei, calcareous parts,cartilaginous tissues, bacteria and mucus in livid to light blue andeosin stains cytoplasm, interstitial tissues, various fibers,erythrocytes and keratinocyte in red to dark red. In other staining formorphological observation, cell nuclei, calcareous parts, cartilaginoustissues, bacteria and mucus may be stained in livid to light blue with ahematoxylin analogue or a dye having an absorption wavelength similar tothat of hematoxylin, and cytoplasm, interstitial tissues, variousfibers, erythrocytes and keratinocyte may be stained in red to dark redwith an eosin analogue or a dye having absorption and emissionwavelengths similar to those of eosin.

[Immunostaining Step]

In the present invention, as an immunostaining method, a fluorescentstaining method in which a biological substance to be detected isstained with the above-described fluorescent label for immunostaining isemployed.

For example, when immunostaining a specific antigen, a method in which alabel (conjugate) is prepared by directly binding a fluorescent labeland a primary antibody and an antigen is then stained (primary antibodymethod), a method in which a label is prepared by directly binding afluorescent label and a secondary antibody and an antigen bound with aprimary antibody is then stained (secondary antibody method) or a methodin which a label is prepared by directly binding a fluorescent label andbiotin and an antigen bound with a primary antibody and avidin or astreptavidin-modified secondary antibody is then stained (biotin-avidinmethod or sandwich method) can be used.

Any primary antibody may be used in the immunostaining and the primaryantibody is variable depending on the subject to be immunostained. Forexample, when immunostaining is performed using HER2 as an antigen, ananti-HER2 antibody is used. In addition, any secondary antibody may beused and the secondary antibody is variable depending on the primaryantibody. Examples of secondary antibody include anti-mouse, rabbit,bovine, goat, sheep, dog and chicken antibodies.

For binding of a fluorescent label with an antibody or biotin, anyexisting method may be used. For example, amidation by reaction betweenamine and carboxylic acid, sulfidation by reaction between maleimide andthiol, imination by reaction between aldehyde and amine, or amination byreaction between epoxy and amine can be used.

The above-described immunostaining is not restricted to tissue staining,and cell staining can be also applied. Further, the biological substanceto be detected is not particularly restricted as long as there is asubstance which specifically binds thereto. Typically, a combination ofan antigen and an antibody is used as described above; however, it isalso possible to use, for example, a combination of a nucleic acidmolecule (oligonucleotide or polynucleotide) and a nucleic acid moleculehaving a sequence hybridizable thereto.

[Fluorescence Observation Step]

By irradiating the pathological specimen subjected to immunostaining andstaining for morphological observation in the above-described steps withan excitation light having a wavelength appropriate for the fluorescentlabel used, the fluorescence emitted by the fluorescent label isobserved. By this step, a prescribed biomolecule existing in thepathological specimen can be detected and this information can beutilized to determine the appropriateness of applying an antibodypharmaceutical (such as Herceptin which targets HER2).

For the irradiation of an excitation light, the same irradiation meansas the one used in an ordinary fluorescence observation may be employed.For example, from a laser light source installed in a fluorescencemicroscope, an excitation light having an appropriate wavelength andoutput may be irradiated to the stained pathological specimen using, asrequired, a filter which selectively allows a light of a prescribedwavelength to pass therethrough.

Observation of fluorescence may be performed either through the lensbarrel of a fluorescence microscope or on a separate display means (suchas a monitor) showing an image taken by a camera mounted on afluorescence microscope. Though depending on the fluorescent substance,even when the fluorescence cannot be sufficiently observed visuallythrough the lens barrel of a fluorescence microscope, the fluorescencemay be observed by taking an image thereof with a camera in some cases.As required, a filter which selectively allows a light of a prescribedwavelength to pass therethrough may also be used.

In the present invention, immunostaining and staining for morphologicalobservation are both performed on the same pathological specimen;however, for observation of an image produced by the staining formorphological observation, it is not required to irradiate thepathological specimen with an excitation light for exciting thefluorescent label used in the immunostaining and the image may beobserved under the same observation conditions as those of a lightmicroscope.

In the biological substance detection method according to the presentinvention, since the brightness retention rate of an immunostained partis in a specific range of ratio with respect to the brightness retentionrate of a part stained for morphological observation, there is noadverse effect on the assessment of the immunostaining results.Therefore, the fluorescence observation can be performed afterirradiating an excitation light for an arbitrary time; however, thefluorescence observation is performed preferably within 90 minutes afterinitiating the irradiation of an excitation light, more preferablywithin 30 minutes after initiating the irradiation of an excitationlight, under normal irradiation conditions (such as irradiation energy).

In the present invention, the ratio (RS/RN) of the brightness retentionrate of an immunostained part (RS) to the brightness retention rate of apart stained for morphological observation (RN) is set to be in a rangeof 80 to 120%, preferably 100 to 120%. When the above-described ratio islower than 80%, the brightness of the immunostained part becomesexcessively low relative to that of the part stained for morphologicalobservation at the time of fluorescence observation, while when theabove-described ratio is higher than 120%, the brightness of the partstained for morphological observation becomes excessively low relativeto that of the immunostained part at the time of fluorescenceobservation. In both of these cases, the information obtained from thestained pathological specimen may be largely degraded by irradiationwith an excitation light.

Here, the brightness of an immunostained part (S) is a value obtained asan average brightness of the sites stained with a fluorescent label forimmunostaining. Further, the brightness retention rate of animmunostained part (RS) is a value which is calculated by an equation,S_(A)/S_(B), from the brightness measured before irradiating anexcitation light for fluorescence observation (S_(B)) and the brightnessmeasured at the time of observing the fluorescence emitted by thefluorescent label after irradiating an excitation light for a certainperiod of time (S_(A)).

Meanwhile, the brightness of a part stained for morphologicalobservation (N) is a value obtained as an average brightness of thesites that are not stained with a fluorescent label for immunostainingbut with a staining agent for morphological observation. Further, thebrightness retention rate of a part stained for morphologicalobservation (RN) is a value which is calculated by an equation,N_(A)/N_(B), from the brightness measured before irradiating anexcitation light for fluorescence observation (N_(B)) and the brightnessmeasured at the time of observing the fluorescence emitted by thefluorescent label after irradiating an excitation light for a certainperiod of time (N_(A)).

The brightness of an immunostained part (S) and that of a part stainedfor morphological observation (N) can be determined by, for example,taking an image using a camera mounted on a fluorescence microscope andthen calculating the brightness of each pixel within a prescribed areaof the image using an image analysis software.

In the fluorescence observation, for example, the ratio of thebrightness retention rates (RS/RN) may be calculated in theabove-described manner for a test sample under certain stainingconditions (combination of a staining agent for morphologicalobservation and a fluorescent label for immunostaining) and excitationlight irradiation conditions (e.g., wavelength and intensity). If theabove-described ratio was confirmed to be in a range of 80 to 120%, thebiological substance detection method can be carried out with a certainlevel of reliability by observing the fluorescence for otherpathological specimen under the same staining and excitation lightirradiation conditions as those used for the test sample. Further, ifthe above-described ratio was found to be outside the range of 80 to120%, the observation of fluorescence for other pathological specimencan be performed after changing the staining and excitation lightirradiation conditions to adjust the ratio to be in a range of 80 to120%.

Alternatively, when the ratio of the brightness retention rates (RS/RN)under particular staining and excitation light irradiation conditionswas once confirmed to be in a range of 80 to 120% as described above, aslong as the same combination and observation conditions are applied, thebiological substance detection method can be assumed to have a certainlevel of reliability and thus be operated without verifying it using thetest sample for every observation.

EXAMPLES

The present invention will now be described in detail by way of examplesthereof; however, the present invention is not restricted to thefollowing examples.

[Sample Preparation] (Sample 1-1: Texas Red-containing Silica Particle)

In DMF, 3.4 mg of a fluorescent dye, Sulforhodamine 101 acid chloride(manufactured by Dojinsha Co., Ltd., Texas Red dye), and 3 μL of3-aminopropyltrimethoxysilane (manufactured by Shin-Etsu Chemical Co.,Ltd., KBM903) were mixed to obtain an organoalkoxysilane compound. Then,0.6 mL of the thus obtained organoalkoxysilane compound was mixed for 3hours with 48 mL of ethanol, 0.6 mL of TEOS (tetraethoxysilane), 2 mL ofwater and 2 mL of 28% aqueous ammonia. The mixture produced in theabove-described step was centrifuged at 10,000 G for 20 minutes and theresulting supernatant was removed. Then, ethanol was added to dispersethe precipitates and the resultant was centrifuged again. Theprecipitates were washed twice with each of ethanol and pure water bythe same procedure. By SEM observation of the thus obtained tetramethylrhodamine-containing silica nanoparticles, the average particle size andthe variation coefficient were found to be 104 nm and 12%, respectively.

The thus obtained fluorescent substance-containing silica nanoparticleswere adjusted with PBS (phosphate-buffered physiological saline)containing 2 mM of EDTA (ethylenediamine tetraacetic acid) to aconcentration of 3 nM. This solution was then mixed with SM (PEG) 12(manufactured by Thermo Fisher Scientific K.K.,succinimidyl-[(N-maleimidopropionamido)-dodecaethylene glycol]ester) toa final concentration of 10 mM and allowed to react for 1 hour. The thusobtained mixture was centrifuged at 10,000 G for 20 minutes and theresulting supernatant was removed. Then, PBS containing 2 mM of EDTA wasadded to disperse the precipitates and the resultant was centrifugedagain. The precipitates were washed three times by the same procedure toobtain fluorescent dye-containing particles for antibody binding.

On another front, anti-human HER 2 antibodies were subjected to areduction treatment with 1M dithiothreitol (DTT) and excess DTT was thenremoved using a gel filtration column, thereby obtaining a solution ofreduced antibodies capable of binding to silica particles.

The thus obtained fluorescent dye-containing particles for antibodybinding and reduced antibodies were mixed in PBS containing 2 mM of EDTAand allowed to react for 1 hour. Thereafter, the reaction was terminatedwith an addition of 10 mM mercaptoethanol. The thus obtained solutionwas centrifuged at 10,000 G for 20 minutes and the resulting supernatantwas removed. Then, PBS containing 2 mM of EDTA was added to disperse theprecipitates and the resultant was centrifuged again. The precipitateswere washed three times by the same procedure to obtain fluorescentdye-containing particles bound with anti-human ER antibody.

(Sample 1-2: ATTO 590-containing Silica Particle)

Fluorescent dye-containing particles bound with anti-human HER2 antibodywere synthesized in the same manner as the sample 1-1, except that ATTO590 dye (manufactured by ATTO-TEC GmbH) was used as the fluorescent dye.

(Sample 1-3: Cresyl Violet-containing Silica Particle)

Fluorescent dye-containing particles bound with anti-human HER2 antibodywere synthesized in the same manner as the sample 1-1, except thatCresyl Violet (manufactured by Sigma-Aldrich) was used as thefluorescent dye and 3-glycidyloxypropyltrimethoxysilane (manufactured byTCI Co., Ltd.) was used in place of 3-aminopropyltrimethoxysilane(manufactured by Shin-Etsu Chemical Co., Ltd., KBM903).

(Sample 1-4: Perylene Diimide-containing Silica Particle)

Perylene diimide, which was used as a fluorescent dye, was prepared bythe following method.N,N′-bis(2,6-diisopropylphenyl)-1,6,7,12-tetraphenoxyperylene-3,4:9,10-tetracarboxdiimidewas treated with concentrated sulfuric acid to prepare a perylenediimide sulfonic acid derivative. This was then converted to an acidchloride to obtain a perylene diimide sulfonic acid chloride derivative.Fluorescent dye-containing particles bound with anti-human HER2 antibodywere synthesized in the same manner as the sample 1-1, except that thethus obtained perylene diimide sulfonic acid chloride derivative wasused as the fluorescent dye.

(Sample 1-5: Texas Red-containing Melamine Particle)

After adding 2.5 mg Sulforhodamine 101 (manufactured by Sigma-Aldrich)to 22.5 mL of water, the resultant was heated at 70° C. for 20 minuteson a hot stirrer, followed by an addition of 1.5 g of water-solublemelamine resin “Nikalac MX-035” (manufactured by Nippon CarbideIndustries Co., Ltd.) and heating with stirring for another 5 minutes.Then, 100 μL of formic acid was added and the resultant was heated withstirring for 20 minutes at 60° C. and subsequently cooled to roomtemperature. Thereafter, the resulting reaction mixture was placed in acentrifugal tube and centrifuged using a centrifuge at 12,000 rpm for 20minutes, followed by removal of the resulting supernatant. Theprecipitates were washed with ethanol and water. After modifying thethus obtained particles with SM(PEG)12 (manufactured by Thermo FisherScientific K.K., succinimidyl-[(N-maleimidopropionamido)-dodecaethyleneglycol] ester) in the same manner as in the case of the sample 1-1,fluorescent dye-containing particles bound with anti-human HER2 antibodywere synthesized.

(Sample 1-6: ATTO 590-containing Melamine Particle)

Fluorescent dye-containing particles bound with anti-human HER2 antibodywere synthesized in the same manner as the sample 1-5, except that ATTO590 (manufactured by ATTO-TEC GmbH) was used as the fluorescent dye.

(Sample 1-7: Perylene Diimide-containing Melamine Particle)

Fluorescent dye-containing particles bound with anti-human HER2 antibodywere synthesized in the same manner as the sample 1-5, except that aperylene diimide sulfonic acid derivative was used as the fluorescentdye.

(Sample 1-8: Texas Red-containing Cross-linked PS Particle)

Texas Red dye-containing cross-linked PS particles were prepared by asoap-free emulsion polymerization method. A fluorescent dye,Sulforhodamine 101 acid chloride (manufactured by Dojinsha Co., Ltd.,Texas Red dye), was mixed with 4-aminostyrene (manufactured by TokyoChemical Industry Co., Ltd.) at room temperature for 1 hour to preparedye-bound styrene. To 5 mL of pure water aerated by argon bubbling, 0.18g of glycidyl methacrylate (manufactured by Tokyo Chemical Industry Co.,Ltd.), 0.05 g of styrene (manufactured by Wako Pure Chemical Industries,Ltd.), 0.05 g of divinylbenzene and 0. 005 g of the thus obtaineddye-bound styrene were added. After heating the resultant with stirringto 70° C., 0.012 g of a water-soluble azo polymerization initiator, V-50(manufactured by Wako Pure Chemical Industries, Ltd.), was added and theresulting mixture was allowed to react for 12 hours. The resultingreaction solution was centrifuged at 10,000 G for 20 minutes to recoverparticles. The recovered particles were purified by dispersing them inpure water and then centrifuging the resulting dispersion once again forrecovery. The thus obtained particles were added to an excess amount ofaqueous ammonia so as to convert the epoxy groups at the particleterminals into amino groups. After modifying the resulting particleswith SM(PEG)12 (manufactured by Thermo Fisher Scientific K.K.,succinimidyl-[(N-maleimidopropionamido)-dodecaethylene glycol] ester) inthe same manner as in the case of the sample 1-1, fluorescentdye-containing particles bound with anti-human HER2 antibody weresynthesized.

(Sample 1-9: ATTO 590-containing Cross-linked PS Particle)

Fluorescent dye-containing particles bound with anti-human HER2 antibodywere synthesized in the same manner as the sample 1-8, except that ATTO590 (manufactured by ATTO-TEC GmbH) was used as the fluorescent dye.

(Sample 1-10: Cresyl Violet-containing Cross-linked PS Particle)

Fluorescent dye-containing particles bound with anti-human HER2 antibodywere synthesized in the same manner as the sample 1-8, except thatCresyl Violet (manufactured by Sigma-Aldrich) was used as thefluorescent dye.

(Sample 1-11: Perylene Diimide-containing Cross-linked PS Particle)

Fluorescent dye-containing particles bound with anti-human HER2 antibodywere synthesized in the same manner as the sample 1-8, except that aperylene diimide sulfonic acid derivative converted into an acidchloride was used as the fluorescent dye.

(Sample 2-1: Cy 3.5-containing Silica Particle)

Fluorescent dye-containing particles bound with anti-human HER2 antibodywere synthesized in the same manner as the sample 1-1, except thatCy-3.5 SE (manufactured by Roche) was used as the fluorescent dye.

(Sample 2-2: Cy 3.5-containing Melamine Particle)

Fluorescent dye-containing particles bound with anti-human HER2 antibodywere synthesized in the same manner as the sample 1-5, except thatCy-3.5 SE (manufactured by Roche) was used as the fluorescent dye.

(Sample 2-3: BODIPY TR-containing Silica Particle)

Fluorescent dye-containing particles bound with anti-human HER2 antibodywere synthesized in the same manner as the sample 1-1, except thatBODIPY TR (manufactured by Invitrogen) was used as the fluorescent dye.

(Sample 2-4: BODIPY TR-containing Melamine Particle)

Fluorescent dye-containing particles bound with anti-human HER2 antibodywere synthesized in the same manner as the sample 1-5, except thatBODIPY TR (manufactured by Invitrogen) was used as the fluorescent dye.

[Evaluation by Tissue Staining]

Using the samples 1-1 to 1-11 and 2-1 to 2-4, immunostaining andstaining for morphological observation (HE staining) were performed onhuman breast tissue. The samples 1-1 to 1-11 correspond to Examples 1 to11 and the samples 2-1 to 2-4 correspond to Comparative Examples 1 to 4.

As a section to be stained, a tissue array slide manufactured by CosmoBio Co., Ltd. (CB-A712) was used. After subjecting the tissue arrayslide to a deparaffinization treatment, an antigen inactivationtreatment was performed by subjecting the tissue array slide todisplacement washing with water and a 15-minute autoclave treatment in10 mM citrate buffer (pH 6.0). After the antigen inactivation treatment,the tissue array slide was washed with a PBS buffer and then subjectedto a blocking treatment with a 1% BSA-containing PBS buffer for 1 hourin a moist chamber. Thereafter, the tissue section was allowed to reactfor 3 hours with each of the samples 1-1 to 1-11 and 2-1 to 2-4 thatwere diluted with a 1% BSA-containing PBS buffer to a concentration of0.05 nM. After the reaction with each of the samples 1-1 to 1-11 and 2-1to 2-4, the resulting tissue array slide was washed with a PBS buffer.

After the immunostaining, staining for morphological observation (HEstaining) was performed. The immunostained section was stained withhematoxylin for 5 minutes in Mayer's hematoxylin solution and thenwashed with running water (about 45° C.) for 3 minutes. Then, afterstaining the section with eosin for 5 minutes in a 1% eosin solution, anoperation of immersing the resulting section in pure ethanol for 5minutes was repeated four times to perform washing and dehydration.Subsequently, an operation of immersing the section in xylene for 5minutes was repeated four times to perform clearing. Lastly, the sectionwas sealed with a sealant, Entellan New (manufactured by Merck KGaA),thereby obtaining a sample slide for observation.

The respective tissue sections that were immunostained with the samples1-1 to 1-11 and 2-1 to 2-4 and then stained for morphologicalobservation were allowed to emit fluorescence by irradiation with anexcitation light. Then, using an inverted fluorescence microscope(manufactured by Carl Zeiss), images were obtained from the respectivetissue sections.

Using an optical filter, the excitation and fluorescence wavelengths(nm) were set to be 575 to 600 nm and 612 to 682 nm, respectively(Tables 1 and 2 show the maximum absorption and maximum emissionwavelengths of the particles). The conditions of the excitationwavelength in the microscope observation and image acquisition were setsuch that the irradiation energy around the center of the field of viewbecame 900 W/cm² for 580-nm excitation and 500 W/cm² for 365-nmexcitation. In the image acquisition, the exposure time was arbitrarilyset such that the image brightness was not saturated. The measurementswere performed at an exposure time of, for example, 4,000 μs.

The brightness of each pixel was calculated from the thus obtainedrespective images using an image analysis software, Image-J, and theaverage brightness of the sites that were stained with each fluorescentlabel of the samples 1-1 to 1-11 and 2-1 to 2-4 (immunostained part) wascalculated (brightness of the immunostained part). This averagebrightness corresponds to a signal value (S). A brightness of “0” wasdefined as black (darkest) and a brightness of “255” was defined aswhite (brightest). At the same time, the average brightness of the sitesthat were not stained with the fluorescent label but with eosin in thevicinity of the fluorescently-labeled cells (part stained formorphological observation) was also calculated (brightness of the partstained for morphological observation). This average brightnesscorresponds to a noise value (N). The ratio of the brightness ofimmunostained part and that of part stained for morphologicalobservation was defined as “S/N ratio”.

Decoloration of the immunostained part and the part stained formorphological observation was evaluated by continuing irradiation of anexcitation light with the field of view being fixed for 30 minutes,determining the brightness of the immunostained part and that of thepart stained for immunological observation from both microscope imagesthat were taken prior to the irradiation (at the beginning of theirradiation, 0 minute) and after the irradiation (30 minutes), and thencalculating the brightness retention rate, which is represented by anequation: (brightness after 30-minute irradiation/brightness immediatelyafter the initiation of irradiation), for both of the immunostained partand the part stained for immunological observation. In addition, thepre-irradiation and post-irradiation S/N ratios were calculated todetermine the rate of change in the S/N ratio before and after theirradiation, which is represented by an equation: (post-irradiation S/Nratio—pre-irradiation S/N ratio)/pre-irradiation S/N ratio. This valuewas used as an index of the difference in the appearance before andafter the irradiation with an excitation light.

Further, a serial section composed of the sections simultaneouslysubjected to the above-described immunostaining and staining formorphological observation was stained with DAB to set in advance aregion in which the expression of HER2 on cell membrane was limited(1+). A region corresponding thereto of a section simultaneouslysubjected to immunostaining and staining for morphological observationwas visually evaluated before and after the irradiation under afluorescence microscope. When the evaluation did not change before andafter the irradiation, the section was assigned with “∘”, and when theevaluation changed before and after the irradiation, the section wasassigned with “×”.

TABLE 1 Example1 Example2 Example3 Example4 Example5 Example6 0 min. 30min. 0 min. 30 min. 0 min. 30 min. 0 min. 30 min. 0 min. 30 min. 0 min.30 min. Dye Texas Red ATTO590 Cresyl violet Perylene Texas Red ATTO590Parent material Silica Silica Silica Silica Melamine Melamine Maximumabsorption 585 594 600 580 590 595 wavelength (nm) Maximum emission 613624 620 620 612 624 wavelength (nm) Brightness of eosin-stained 20 16 2016 20 16 20 16 20 16 20 16 part Brightness of 80 59 80 60 80 52 80 71 8058 80 58 fluorescently-stained part S/N ratio 4.0 3.7 4.0 3.8 4.0 3.34.0 4.4 4.0 3.6 4.0 3.6 Rate of change in S/N ratio −8 −5 −18 10 −10 −10before and after irradiation (%) Brightness retention rate of 80 80 8080 80 80 eosin-stained part after 30 minutes in relation to thebrightness at 0 minute (%) Brightness retention rate of 74 75 66 89 7372 fluorescently-stained part after 30 minutes in relation to thebrightness at 0 minute (%) Ratio of the brightness 92 94 82 111 91 90retention rate of fluorescently-stained part to the brightness retentionrate of eosin-stained part (%) Concordance between the ∘ ∘ ∘ ∘ ∘ ∘visual evaluation results obtained at 0 and 30 minutes Example 7 Example8 Example 9 Example 10 Example 11 0 min. 30 min. 0 min. 30 min. 0 min.30 min. 0 min. 30 min. 0 min. 30 min. Dye Perylene Texas Red ATTO590Cresyl Violet Perylene Parent material Melamine Cross-linked PSCross-linked PS Cross-linked PS Cross-linked PS Maximum absorption 580580 594 600 580 wavelength (nm) Maximum emission 625 615 624 620 620wavelength (nm) Brightness of eosin-stained 20 16 20 16 20 16 20 16 2016 part Brightness of 80 72 80 64 80 68 80 67 80 77fluorescently-stained part S/N ratio 4.0 4.5 4.0 4.0 4.0 4.2 4.0 4.2 4.04.8 Rate of change in S/N ratio 13 0 5 5 20 before and after irradiation(%) Brightness retention rate of 80 80 80 80 80 eosin-stained part after30 minutes in relation to the brightness at 0 minute (%) Brightnessretention rate of 90 80 85 83 96 fluorescently-stained part after 30minutes in relation to the brightness at 0 minute (%) Ratio of thebrightness 112 100 106 104 120 retention rate of fluorescently-stainedpart to the brightness retention rate of eosin-stained part (%)Concordance between the ∘ ∘ ∘ ∘ ∘ visual evaluation results obtained at0 and 30 minutes Comparative Comparative Comparative Comparative Example1 Example 2 Example 3 Example 4 0 min. 30 min. 0 min. 30 min. 0 min. 30min. 0 min. 30 min. Dye cy3.5 cy3.5 BODIPY TR BODIPY TR Parent materialSilica Melamine Silica Melamine Maximum absorption 581 590 588 592wavelength (nm) Maximum emission 596 608 616 620 wavelength (nm)Brightness of eosin-stained 20 16 20 16 20 16 20 16 part Brightness of80 44 80 42 80 49 80 47 fluorescently-stained part S/N ratio 4.0 2.7 4.02.6 4.0 3.1 4.0 3.0 Rate of change in S/N ratio −33 −35 −23 −25 beforeand after irradiation (%) Brightness retention rate of 80 80 80 80eosin-stained part after 30 minutes in relation to the brightness at 0minute (%) Brightness retention rate of 54 53 62 59fluorescently-stained part after 30 minutes in relation to thebrightness at 0 minute (%) Ratio of the brightness 68 66 77 74 retentionrate of fluorescently-stained part to the brightness retention rate ofeosin-stained part (%) Concordance between the x x x x visual evaluationresults obtained at 0 and 30 minutes

In Table 1 and FIG. 2, Examples 1 to 11 correspond to the samples 1-1 to1-11 and Comparative Examples 1 to 4 correspond to the samples 2-1 to2-4. In each of Examples and Comparative Examples, the dye and parentmaterial of the particles were different and the rate of the brightnesschange after the irradiation with an excitation light was alsodifferent. Consequently, the S/N ratio, which is the ratio of thebrightness of the immunostained part in relation to that of the partstained for morphological observation, also changed before and after theirradiation with an excitation light. In Examples where the brightnessretention rate of the immunostained part was in a range of 80 to 120% inrelation to the brightness retention rate of the part stained formorphological observation, the region in which the expression of HER2 oncell membrane was limited (1+) was given the same evaluation before andafter the irradiation with an excitation light and the results show thatthere was no profound effect on the appearance of the observationsamples. On the other hand, in Comparative Examples where the brightnessretention rate of the immunostained part was outside the range of 80 to120% in relation to the brightness retention rate of the part stainedfor morphological observation, different evaluations were given beforeand after the irradiation with an excitation light and the results showthat there was an effect on the appearance of the observation samples.

From the above, it is seen that, when staining for morphologicalobservation and immunostaining are simultaneously performed, even if thefluorescent label and/or the staining agent is/are deteriorated, aproper judgment can be made as long as the brightness retention rate ofan immunostained part is in a range of 80 to 120% in relation to thebrightness retention rate of a part stained for morphologicalobservation when the fluorescent label used in the immunostaining isobserved.

1. A biological substance detection method for detecting a biologicalsubstance from a pathological specimen, said method comprising: (a)performing on a test sample an immunostaining step (1a′) using afluorescent label and a staining step (1b′) using a staining agent formorphological observation to form a part of the test sampleimmunostained with the fluorescent label and a part of the test samplemorphologically stained with the staining agent for morphologicalobservation; (b) performing on the immunostained and morphologicallystained test sample a fluorescence observation step (2a′) by irradiatingthe immunostained and morphologically stained test sample with anexcitation light having a wavelength that induces a fluorescent emissionfrom the fluorescent label, simultaneously measuring a brightness of theimmunostained part (S) and a brightness of the morphologically stainedpart (N) each at a time of beginning the irradiation and at a time afterthe irradiation for a certain period of time, obtaining a brightnessretention rate of the immunostained part (RS) and a brightness retentionrate of the morphologically stained part (RN), and calculating a ratioof the brightness retention rates (RS/RN), whereinRS=S _(A) /S _(B), S_(A)=brightness of the immunostained part (S) at thetime after the irradiation for a certain period of time,S_(B)=brightness of the immunostained part (S) at the time of beginningthe irradiation,RN=N _(A) /N _(B), N_(A)=brightness of the morphologically stained part(N) at the time after the irradiation for a certain period of time,N_(B)=brightness of the morphologically stained part (N) at the time ofbeginning the irradiation; (c) selecting a combination of thefluorescent label and the staining agent having the ratio (RS/RN) in therange of 80% to 120% on a basis of a result of the test sample performedin the immunostaining step (1a) and the morphologically staining step(1b) for morphological observation on the test sample; (d) performing ona pathological specimen an immunostaining step (1a) and amorphologically staining step (1b) for morphological observation usingthe selected combination of the fluorescent label and the staining agentto form a part of the pathological specimen immunostained with thefluorescent label and a part of the pathological specimenmorphologically stained with the staining agent for morphologicalobservation; and (e) performing on the immunostained and morphologicallystained pathological specimen a fluorescence observation step (2a) byirradiating the immunostained and morphologically stained pathologicalspecimen with an excitation light having a wavelength that induces afluorescent emission from the fluorescent label and a morphologicalobservation step (2b) without irradiating the excitation light, so as todetect the biological substance from the pathological specimen used inthe step (c), wherein the fluorescent label is a fluorescentdye-containing nanoparticle, said fluorescent dye being selected fromthe group consisting of a rhodamine-based dye molecule, asquarylium-based dye molecule, a cyanine-based dye molecule, an aromaticring-based dye molecules, an oxazine-based dye molecule, acarbopyronine-based dye molecule and a pyrromethene-based dye molecule,and the fluorescence dye has a maximum excitation wavelength in awavelength range of 550 to 620 nm and a maximum emission wavelength in awavelength range of 580 to 700 nm, and wherein the staining agent formorphological observation has a maximum excitation wavelength in awavelength range of 450 to 550 nm.
 2. The biological substance detectionmethod of claim 1, wherein the fluorescent dye is the rhodamine-baseddye molecule or the aromatic ring-based dye molecule.
 3. The biologicalsubstance detection method of claim 2, wherein the fluorescent dye isperylene.
 4. The biological substance detection method of claim 1,wherein the staining agent is eosin.
 5. The biological substancedetection method of claim 1, wherein a parent material of thefluorescent dye-containing nanoparticle is an organic material.
 6. Thebiological substance detection method of claim 5, wherein the parentmaterial of the fluorescent dye-containing nanoparticle is selected fromthe group consisting of polystyrene, polyamide, polylactic acid,polyacrylonitrile, polyglycidyl methacrylate, polymelamine, polyurea,polybenzoguanamine, polyfuran, polyxylene, phenol resin, andpolysaccharide.
 7. The biological substance detection method of claim 1,further comprising, after the immunostaining step (1a) and themorphologically staining step (1b) and prior to the fluorescenceobservation step (2a) and the morphological observation step (2b),performing on the immunostained and morphologically stained pathologicalspecimen a dehydration step, a clearing step, and a sealing step with asealing agent comprising an organic solvent.
 8. A method for screening acombination of a fluorescent label and a staining agent formorphological observation, comprising: performing on a test sample animmunostaining step (1a′) using the fluorescent label and a stainingstep (1b′) using the staining agent for morphological observation toform a part of the test sample immunostained with the fluorescent labeland a part of the test sample morphologically stained with the stainingagent for morphological observation; performing on the immunostained andmorphologically stained test sample a fluorescence observation step(2a′) by irradiating the immunostained and morphologically stained testsample with an excitation light having a wavelength that induces afluorescent emission from the fluorescent label and simultaneouslymeasuring a brightness of the immunostained part (S) and a brightness ofthe morphologically stained part (N) each at a time of beginning theirradiation and at a time after the irradiation for a certain period oftime, obtaining a brightness retention rate of the immunostained part(RS) and a brightness retention rate of the stained part (RN), andcalculating a ratio of the brightness retention rates (RS/RN), whereinRS=S _(A) /S _(B), S_(A)=brightness of the immunostained part (S) at thetime after the irradiation for a certain period of time,S_(B)=brightness of the immunostained part (S) at the time of beginningthe irradiation,RN=N _(A) /N _(B), N_(A)=brightness of the morphologically stained part(N) at the time after the irradiation for a certain period of time,N_(B)=brightness of the morphologically stained part (N) at the time ofbeginning the irradiation; and selecting the combination of thefluorescent label and the staining agent having a ratio of thebrightness retention rates (RS/RN) in a range of 80% to 120%.